The present invention relates generally to systems and methods for processing data representing sigmoid-type or growth curves such as Polymerase Chain Reaction (PCR) curves, and more particularly to systems and methods for determining cross-talk characteristics of PCR detection systems.
The Polymerase Chain Reaction (PCR) is an in vitro method for enzymatically synthesizing or amplifying defined nucleic acid sequences. The reaction typically uses two oligonucleotide primers that hybridize to opposite strands and flank a template or target DNA sequence that is to be amplified. Elongation of the primers is catalyzed by a heat-stable DNA polymerase. A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in an exponential accumulation of a specific DNA fragment. Fluorescent probes are typically used in the process to facilitate detection and quantification of the amplification process.
A set of typical real-time PCR curves is shown in FIG. 1a, where fluorescence intensity values are plotted vs. cycle number for a typical PCR process. In this case, the formation of PCR products is monitored in each cycle of the PCR process. The amplification is usually measured in thermocyclers which include components and devices for measuring fluorescence signals during the amplification reaction. An example of such a thermocycler is the Roche Diagnostics LightCycler (Cat. No. 20110468). The amplification products are, for example, detected by means of fluorescent labelled hybridization probes which emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA. As can be seen in FIG. 1a, the PCR curves include a baseline region 5 and a plateau region 6. The region between the baseline region 5 and the plateau region 6 is typically referred to as the growth region.
Typical PCR detection systems for analyzing radiation emissions from PCR experiments include two or more filters that are each operable to isolate a wavelength range for further analysis. For example, each optical filter typically allows substantially all radiation in a defined wavelength range to pass. However, the probes or markers typically emit with partially overlapping wavelength bands, and a filter's band pass typically includes a region of this overlap such that each detection channel will typically receive signal emitted from other probes. Such cross-talk signals tend to affect the real signal of interest. Accordingly, it is desirable to correct for such cross-talk signals in each detection channel. One traditional way of doing this is to determine quantitative cross-talk coefficients that can be used to correct for cross-talk signals in each detection channel.
In current cross-talk methodologies, the cross-talk coefficients are typically calculated using a ratio of the average plateau values of a basis and cross-talk signal; conventional methods rely exclusively on the plateau region which contains less than 10% of the data. Also, during PCR, the plateau region signal is generated when the chemistry is in an unstable state. For this reason, a baseline signal threshold is typically employed for target identification. Therefore, the conventional methods use a noisy signal to determine cross-talk coefficients with limited information that does not include data from the true signal acquisition region on the curve. Further, incorrect assumptions used in conventional crosstalk models have also been found to induce errors as a function of the data acquisition curve. Thus, the conventional method of calculating cross-talk coefficients may be satisfactory providing that (1) a plateau exists, (2) the plateau is flat, and (3) there is minimum noise in the plateau. However, there are many data sets where this will not be the case.
It is therefore desirable to provide systems and methods for determining cross-talk coefficients in curves, such as sigmoid-type or growth curves, and PCR curves in particular, which overcome the above and other problems.